Background Ruxolitinib (RUX) has been recently approved for the therapy of patients (pts) with myelofibrosis (MF). Although various definitions for response and resistance to this drug have been proposed in the setting of clinical trials, none of them has been validated in clinical practice. Based on RUX efficacy, it is assumed that primary resistance is related to absence or minor reduction in spleen size and constitutional symptoms, while spleen regrowth and recurrence of symptoms after a period with good response establish secondary resistance. The extents of all these parameters have not been clearly defined yet for patients’ management in clinical practice. On the other hand, several biological mechanisms of resistance have been described. In particular, acquisition of new mutations in the predicted RUX-binding region (like Y931C, G935R, R938L, I960V and E985K) was previously shown to confer resistance to JAK inhibitors in vitro (Hornakova et al, Haematologica, 2011; Weigert et al, J Exp Med, 2012). Such mutations have not yet been found in pts treated with RUX. Aims 1) Characterize the parameters leading physicians to conclude that RUX-resistance (RUX-R) was reached in a cohort of consecutive MF pts treated with RUX in clinical practice. 2) Identify a molecular signature of RUX-R. Methods Consecutive pts with MF treated with RUX in our center were identified. Clinical characteristics and evolution during and after RUX therapy were collected. Pts were screened for JAK2V617F (PCR), MPL, TET2, and SRSF2 mutations (direct Sanger sequencing). In addition, the last exons of JAK2 corresponding to the RUX-binding domain were sequenced in RUX-R pts. Results In all, 41 pts received RUX between Nov 2009 and May 2013, including 21 primary, 8 post-PV and 12 post-ET MF, and full data were available in 39 of them for this analysis. Median age was 64 yrs; IPSS risk score was high in 46%, and int-1 or 2 in 54%; 72% were JAK2V617F positive, 10% MPL515-positive. Overall, 16/39 (41%) pts were considered RUX-R by their physician, only 4/16 being primary resistant with Conclusion In this series of consecutive pts treated with RUX in a single center, RUX-R was observed in 41% of pts, mostly as a late event (after median exposure of 1 year) and rarely as primary resistance (10%). RUX-R was always associated with spleen regrowth, and accompanied with recurrence of symptoms in half of the cases. RUX-R was more frequently seen in pts with high risk IPSS score, post-ET MF, smaller spleen response (-50% vs. -80% in non RUX-R), but the only factor significantly associated with RUX-R was the absence of mutation in all of the following genes: JAK2, MPL, TET2, and SRSF2. Furthermore, sequencing of the kinase domain of JAK2 in RUX-R pts didn’t detect any mutation possibly affecting drug binding, suggesting that such mechanism is rarely involved in clinical resistance to RUX. Other mechanisms, like the phenomenon of “persistence” to JAK inhibitor therapy described by Koppikar at al (Nature, 2012) should be investigated in these pts. Disclosures: Robin:Novartis: Research Funding. Rea:Novartis: Honoraria; BMS: Honoraria; Pfizer: Honoraria; Teva: Honoraria; Ariad: Honoraria. Giraudier:NOvartis: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Honoraria. Kiladjian:AOP Orphan: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Sanofi: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Celgene: Research Funding; Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding.